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Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Transcriptional silencing of the gene coding for amoebapore A (AP-A) was observed when trophozoites of Entamoeba histolytica were transfected with a hybrid plasmid construct containing the ap-a gene flanked by the upstream and downstream segments of the original Ehap-a gene. Transfectants were totally devoid of ap-a transcript and AP-A protein. An identical silencing effect was observed upon transfection with a plasmid that contained only the 5 upstream region of ap-a. Removal of the selecting antibiotic enabled the isolation of plasmidless clones, which retained in their progeny the silenced phenotype. E. histolytica cells were able to overexpress ap-a when transfected with a plasmid containing the gene flanked by the 5 and 3 regions of the EhRP-L21 gene. This plasmid, however, could not express ap-a in the retransfected, cloned trophozoites lacking AP-A. This is the first report of gene silencing in E. histolytica, and the mechanism appears to belong to transcriptional gene silencing and not to posttranscriptional gene silencing. This conclusion is based on the following results: (i) silencing was achieved by transfection of homologous 5 flanking sequences (470 bp of the Ehap-a gene), (ii) transcription initiation of Ehap-a was found to be blocked, and (iii) short double-stranded RNA fragments of the ap-a coding and noncoding sequences were not detected. Trophozoites lacking AP-A are nonpathogenic and impaired in their bacteriolytic capability.The amoebapores (AP) are an important virulence factor of Entamoeba histolytica (12,30). Three isoforms of the small (77 amino acid) AP protein exist as mature and potentially active peptides inside distinct cytoplasmic granules of the trophozoite (30-32). The present view of the mode of action of AP is that following lectin-mediated recognition and intimate adherence between the trophozoite and its target cell, the AP molecules are inserted into the membranes of the latter without depending on the interaction with a specific membrane receptor and that antibodies against AP are thus unable to inhibit its toxic effect. To investigate the specific role of AP-A, the most abundant among the three isoforms (38), in the pathogenicity of the parasite, the levels of AP-A expression were modulated by transfection of trophozoites with different hybrid plasmid constructs. Down-regulation (60%) of expression of AP-A by antisense mRNA caused a drastic reduction in amoeba pathogenicity and clearly demonstrated its importance in the parasite's virulence (12). Interestingly, overexpression (fourfold) of AP-A also caused a dramatic reduction in virulence (13). This result has been attributed to an observed spillover of AP-A from the granules into the cytoplasm and a continued release of AP-A by viable trophozoites into the surrounding medium. In an attempt to overcome the problem of the apparent mislocalization of the overexpressed AP-A, we prepared another hybrid plasmid construct in which the Ehap-a gene was inserted into the vector, flanked by its original 5Ј and 3Ј regulatory element...